Effects of bone marrow derived stem cells and PDGF/BMP-2 on the treatment of radiation induced skin ulcer and osteonecrosis in rats

Abstract

Background and Purpose Osteoradionecrosis (ORN) of the mandible is a serious complication of radiation therapy, and it is preceded by soft tissue damage before bone loss appears. ORN is defined as a condition in which irradiated bone becomes exposed through a wound in the overlying skin or mucosa and persists without healing for 3 to 6 months. In the early stage of ORN, soft tissue necrosis occurs and is followed by exposure of bone tissue. As ORN proceeds, not only the exposed cortical bone but also the underlying medullary bone undergoes necrosis, and eventually necrosis progresses to the lower body of the mandible. Because pathology of ORN was not understood clearly, a direct treatment modality has not been established for ORN. Since ORN begins from necrosis of soft tissue, delaying the speed of necrosis or improving the healing potential at this stage would minimize subsequent exposure of bone. After bone exposure, improvement of bony healing potential at the bony necrosis stage would also be beneficial in accelerating treatment of ORN. Recently, various effects of mesenchymal stem cells on wound healing have been reported. However, studies are lacking in the effect of application of mesenchymal stem cells or their combination with growth factors on healing of ORN at each stage. This study investigated the effect of platelet derived growth factor (PDGF) or mesenchymal stem cells (rMSCs) on radiation-induced soft tissue injury and the effect of rat mesenchymal stem cells (rMSCs) or bone morphogenetic protein-2 (BMP-2) on the osseous healing of osteoradionecrosis in the rat mandible, depending on application time. Part I. Effect on radiation induced soft tissue injury [Materials and Methods] Sprague-Dawley rats (n=17) were irradiated on the right and left buttocks with a single dose of 50 Gy. The right buttocks were administered with phosphate-buffered solution as a control. The left buttocks were administered with either rMSCs (2 x 106 cells), PDGF (8 μg), or PDGF combined with rMSCs. Administration was done at three weeks after irradiation. Wound healing was analyzed by calculating the percentage of residual ulcerated skin area compared to the total irradiated area during the five week healing period after administration. Modified skin scores were also assessed. Finally, skin lesions were histologically evaluated. [Results] More than 40% of the irradiated skin area within the irradiated zone underwent ulceration within 16 days postirradiation, with peak ulceration exceeding 50% around three weeks post-irradiation. Administration of rMSCs or PDGF alone did not confer any significant healing effect. The combined rMSCs+PDGF treatment significantly reduced the wound size compared with the nontreated control up to two weeks postinjection. Regarding the histological examination, lesions administered with PDGF (either alone or mixed with rMSCs) resulted in a greater deposition of highly organized collagen fibers throughout the dermis layer, compared with the control. Part II. Effect on radiation induced bony necrosis [Materials and Methods] The mandibles of SD rats were irradiated while sparing the body and internal organs by utilizing a non-occlusive skin clamp along with an x-ray image guided stereotactic irradiator. All wounds were created using the 30 Gy dose level on the right mandible at a 100 cm source-to-surface distance. One week after irradiation, all molar teeth on the right side of the mandible were extracted. Rats were randomly divided into two groups according to time of application of a hydrogel complex (n=25, each group). In Group 1, rMSCs and/or BMP-2 carried with hydrogel was applied immediate after surgery. In Group 2, it was done after the occurrence of ORN, four weeks after surgery. Micro-CT data of bone healing were compared among groups without hydrogel (n=5) and with hydrogel alone (n=5), or mixed with 2 x 104 of rMSCs (n = 5), or 10 ㎍ of BMP-2 (n = 5), or both of them (n = 5). Animals without irradiation were used as the negative control. [Results] In Group 1, BMP-2 was effective in increasing both bone volume (BV) and bone mineral density (BMD), while rMSCs were not. All animals showed ORN at irradiated areas in Group 2. In Group 2, the combined application of rMSCs and BMP-2 significantly increased BMD and BV compared to the groups without hydrogel and with hydrogel alone. BMP-2 application in Group 1 resulted in significantly higher BMD (6.29 ± 1.63%) compared to BMP-2 application in Group 2 (5.71 ± 0.94%). Conclusions The combined administration of rMSCs and PDGF efficiently enhanced the healing of radiation-induced skin ulceration. Osseous healing after post-irradiation trauma in rats was enhanced immediately after dentoalveolar trauma by application of BMP-2, while the combined application of rMSCs and BMP-2 was most effective after osteoradionecrosis occurred. Key